Low cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is greatly hindered by undesirable mutations in the baculovirus genome. Engineering of the virus to overcome such mutations is crucial in enabling continuous large-scale production of baculovirus. Establishment of large scale production of baculovirus can be of great importance in producing baculovirus biopesticide as well as baculovirus-expressed proteins (used as vaccine, diagnostics and other biopharmaceutical products). Few polyhedra (FP) mutants and defective interfering particles (DIP) are commonly responsible for the reduction in occluded virus (polyhedra) yield and decreased infectivity of the virus. FP phenotype commonly results from the insertion of transposons from the host insect cell into the baculovirus fp25k gene. We demonstrated that engineering of the wild type baculovirus fp25k gene (removal of the transposon sites from the fp25k gene) reduced the incidence of the FP mutants in late passages. No evidence of transposon insertion has been found in this recombinant virus (stabilized virus) even at very late passages. Therefore the stabilized virus could be useful for continuous production for a longer period of time than the wild type virus. Moreover, we found the evidence of mutations other than “insertion mutation” contributing significantly in formation of FP phenotype and hence the novel recombinant virus could not eliminate the FP phenotype completely in very late passages. We have developed assays for detection of other mutants/DIPs based on genotypic and phenotypic analysis of late passaged virus population.

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